Anaplasma phagocytophilum is an obligate intracellular bacterium that invades neutrophils to cause the emerging and potentially fatal infection, human granulocytic anaplasmosis. The host cell-derived vacuole in which A. phagocytophilum resides hijacks recycling endosomes, a process that is essential for the bacterium's survival. How this unique pathogen-occupied organelle commandeers endocytic sorting is poorly understood. Monoubiquitination has recently emerged as a posttranslational modification that regulates endocytic sorting, particularly the recycling endosome pathway. Monoubiquitin is itself a sorting signal, and proteins decorated with the moiety make extensive contacts with endocytic machinery. We discovered that the A. phagocytophilum vacuolar membrane accumulates monoubiquitin and that P100, an effector that localizes to the vacuolar membrane, carries three F-boxes. The F-box motif was first identified in eukaryotes and interacts with the SCF ubiquitin ligase complex, which catalyzes ubiquitination of proteins. Monoubiquitin colocalizes with GFP-P100 when it is ectopically expressed in eukaryotic cells and with endogenous P100 on the A. phagocytophilum vacuolar membrane. The P100 region that contains the F-box motif is exposed on the cytosolic face of the A. phagocytophilum vacuolar membrane and competitively inhibits bacterial growth when it is ectopically expressed in infected cells. P100 is a novel bacterial effector because, while many microbial F-box proteins exploit host cell polyubiquitination, P100 is the first example of an F-box effector that co-opts monoubiquitination. Our investigations will test the hypothesis that the P100 F-boxes recruit the SCF ubiquitin ligase complex to direct monoubiquitination of host proteins on the A. phagocytophilum vacuolar membrane and that doing so is important for bacterial growth. In doing so, we will decipher a newly discovered bacterial pathogenic mechanism and will advance knowledge of the strategies by which microbes co-opt ubiquitin pathways to thrive in diverse, even microbiocidal, host cells.